Gonorrhea occurs at high incidence worldwide and has a major impact on reproductive and neonatal health worldwide. Alarmingly, with each new antibiotic introduced for gonorrhea, resistance has emerged, including resistance to penicillin, tetracycline, fluoroquinolones, and recently the third-generation cephalosporins. Treatment options are currently seriously limited and the development of a gonorrhea vaccine is a critical, long- term solution to this problem. Progress on gonorrhea vaccines has been slow, however, in part due to the high number of surface molecules in Neisseria gonorrhoeae (GC) that undergo phase or antigenic variation and a lack of understanding of protective responses. Gonorrhea vaccine development can therefore benefit from a comprehensive, unbiased approach for antigen discovery. We hypothesize that many constituents of the GC cell envelope are stable and important for biological functions, and thus represent attractive vaccine targets. Moreover, there is growing evidence that Th1 responses protect against gonorrhea and we have preliminary data using the vaccine candidate MtrE that supports the effectiveness of Th1-inducing adjuvants in inducing vaccine-mediated protection. Accordingly, here we propose to 1) identify novel conserved vaccine candidates using a proteomics-guided discovery program. High-throughput proteomic studies will be performed to broaden the array of potential antigens that are expressed in response to stimuli encountered at different infection sites including: oxygen availability (aerobic and anaerobic), iron deprivation, and the presence of human serum; 2) evaluate the selected proteins as vaccine candidates. Nineteen protein candidates will be subjected to rigorous evaluation for their potential as vaccine targets by verifying their surface exposure and conservation using a diverse collection of contemporary GC clinical isolates, examining pathophysiological functions in vitro and during experimental murine infection, and by assessing their capacity to elicit functional antibodies in mice; 3) determine the protective capabilities of candidate antigens in a murine genital tract infection model. The three most promising antigens will be tested alone or in combination with each other and with MtrE, to determine their protective potential against GC in the murine infection model. Completion of these proposed studies will provide a starting point for preventive vaccine(s) against GC infections and guide next-generation vaccine design, as well as identify novel proteins that may play a role in GC pathogenesis.